Measuring effect of pH , and of lead nitrate , on the rate of an enzyme reaction Hydrogen peroxide is produced as a by product in many cell

l reactions . ( A by-product is something produced by accident , not something that you want to make . ) Hydrogen peroxide is very harmful to cells so they want to get rid of it as quickly as possible . Cells get rid of hydrogen peroxide using the reaction : 2 H2O2 Hydrogen peroxide 2 H2O Water + O2 ( g ) Oxygen gas

This reaction is catalysed by the enzyme catalase which is found in many cells . Potato extract is a convenient source of catalase . The reaction rate is measured by measuring the volume of oxygen gas produced at different times . Procedure Take the injection syringe out of the socket . Set up the apparatus ( if it has not been done for you ) : Plunger

Injection syringe Vinyl / rubber tube 5.0 cm

3

Gas syringe

Nozzle Needle socket V Bung

Conical flask

so that the vinyl rubber tube is both : i ) straight ( like in the diagram ) and ii ) will not leak gas ( unlike in the diagram ! ) when the bung is in the top of the conical flask . Now take the bung out of the conical flask . Set the gas syringe so that V = 0.00 cm3 . Set the injection syringe at 0.00 cm3 . Measure 10 cm3 5 vol pH7 hydrogen peroxide ( CARE ! GOGGLES ! ) in a 10 cm3 measuring cylinder and put it into the conical flask . If you have a pH meter measure the pH of the hydrogen peroxide solution more accurately . Put the bung in the top of the conical flask so that it will not leak gas . Draw up 5.00 cm3 of potato extract into the injection syringe . Put the injection syringe nozzle into the needle socket BUT DO NOT PUSH THE PLUNGER YET . Get ready to start the stopwatch . Get ready to read the volume V on the gas syringe . Get ready to record the volume V after every 20 seconds in a table : pH 7 Time / s Volume / cm3 WHEN YOU ARE READY push the plunger and start the stopwatch at the same time . ( This might be easiest when you see the potato extract come out of the needle . ) Continue recording until the gas syringe plunger stops moving and so volume V stops changing . ( If everything has worked perfectly the final volume V = 55 cm 3 . If the reaction has given V > 20 cm3 in a reasonable time ( 500 s ? ) then be happy with the results . If the volume V << 20 cm 3 but there were lots of bubbles in a reasonable time then this is a sign that there is a gas leak . Check everywhere where gas might escape , particularly around the bung 2 0 4 0 6 0

and from the 2 ends of the vinyl rubber tubing . Put the apparatus together again with a CLEAN conical flask . Repeat the experiment to see if you now get reasonable results . If the reaction has given very few bubbles very slowly the problem is either the hydrogen peroxide solution or the potato extract . Try repeating the experiment in a CLEAN conical flask with : i ) FRESH potato extract then if it still doesnt work ii ) new hydrogen peroxide solution . If the reaction is too fast to measure then go on to use 20cm 3 , 2.5 vol hydrogen peroxide but continue recording the volumes V until the gas syringe plunger stops moving . ) Once you have a good set of results using 10 cm 3 5.0 vol pH 7 hydrogen peroxide , using a CLEAN conical flask each time , repeat the experiment with the solutions below . There is no need to continue the timing and recording beyond 60 seconds for each experiment . pH = 4 , 10 cm3 , 5 vol pH = 10 , 10 cm3 , 5 vol Processing results Graph : For all your experiments : Construct a new table : pH V20 / cm3 ( V20 5.0 ) / cm3 Initial rate / cm3 s -1

V20 = the volume of gas V produced after 20 seconds in that experiment ( V20 5.0 ) is the volume of oxygen gas actually produced . Remember how you injected 5.0 cm 3 of potato extract and it would be unfair to count that as oxygen gas . Initial rate = ( V20 5.0 ) / 20 because you measured the volume after 20 seconds . Now plot a new graph of initial rate ( y axis ) against pH ( x axis ) Note Vol is a measure of concentration [ ] but the units are not the correct , SI , mol dm -3 . Conclusions

What type of a biochemical is an enzyme ? ________________________ What property of a protein does the action of an enzyme depend on ? ___________________ What holds an enzyme protein in the correct shape ? _________________________________ At what pH was the action of the enzyme best ( fastest ) _______________________________ So at the other pHs what has changed so that the enzyme is not the correct shape to cause its best action ? ___________________________________ Effect of lead nitrate Use the same apparatus and method but this time put : 10 cm3 5 vol pH7 hydrogen peroxide ( CARE ! GOGGLES ! ) cylinder and put it into the conical flask . in a 10 cm3 measuring

And 5 cm3 lead nitrate solution into the flask ( a new syringe ) and swirl the flask to mix the solutions . Then continue to measure the initial rate of the reaction . Time / s Volume / cm3 Look to see what the reaction looks like . What has happened to the rate of the reaction in this experiment ? __________________________ Without the lead nitrate , under these conditions , we would have expected quite a fast reaction . If the rate of reaction with the lead nitrate is faster than without it then do a CONTROL experiment . Do the reaction using 5 cm 3 water instead of the potato extract . if the rate of this control reaction is even faster than with the potato extract enzymes then continue : So what type of a substance is the lead nitrate acting as ?_________________________________ What is the lead ion doing to the enzyme to stop the enzyme acting ? _______________________ 2 0 4 0 6 0